The mucosal surface of the intestinal tract is continuously exposed to innocuous environmental antigens, beneficial commensal microorganisms and potential pathogens. Therefore, maintenance of intestinal immune homeostasis requires a regulated balance between tolerance, immune responsiveness and inflammation. Dysregulation of these processes can result in chronic inflammation or impaired immunity to infection. Intestinal epithelial cells (IECs) provide a physical and biochemical barrier that obstructs entry of commensal and pathogenic bacteria into the underlying lamina propria. In addition, IECs also express pattern recognition receptors and a wide range of immune response genes including cytokines, chemokines and MHC molecules. Therefore, IECs can influence both the composition of commensal communities and host immune responses to them. Recent in vivo studies employing IEC-specific knock-out mice, or the generation of bone marrow chimeric mice in which the non-hematopoietic cell compartment including IECs are specifically targeted, suggest a critical role for IECs in innate recognition of commensal bacteria and regulation of the intestinal immune system. Our preliminary studies suggest two previously unrecognized roles for IECs in the regulation of intestinal immune homeostasis. Metagenomic deep sequence analysis of commensal flora of mice with an IEC-specific deletion of IKK[unreadable] (ikk[unreadable]?IEC) revealed dramatic differences in the composition of commensal bacteria in the intestinal tract of ikk[unreadable]?IEC mice compared to littermate control mice and suggest that IEC-intrinsic NF?B signaling is a critical selective force in influencing the acquisition and/or composition of commensal diversity in health and disease. In addition, deletion of MHC class II exclusively on IECs resulted in dysregulated inflammatory responses following exposure to Citrobacter infection, suggesting an antigen presenting cell function for IECs in immunity to enteric bacterial infection. Based on these preliminary data, two specific aims will (i) test the hypothesis that selective deletion of classical or non-classical NF?B activation in IECs differentially regulates the acquisition and/or stable composition of commensal bacteria in the intestine;and (ii) generate and employ new recombinant Citrobacter strains to test the hypothesis that IEC-intrinsic MHC class II regulates the activation and/or differentiation of bacterial-specific CD4+ T cells in the intestinal microenvironment. Together, the results of these studies will provide new insights into basic mechanisms that govern the relationship between IECs and commensal or pathogenic bacteria, and the consequences of these interactions on homeostasis of immune cell function and host defense in the intestinal microenvironment. It is anticipated that these findings will provide new opportunities to harness the biological functions of IECs in the design of mucosal vaccines and immunotherapeutics. The body's immune system plays a critical role in protecting the exposed surfaces of the body such as the intestine from invading pathogenic bacteria. However, in order to limit chronic inflammatory diseases, the immune system must remain inactive following exposure to beneficial bacteria and environmental antigens. The goals of this proposal are to delineate the mechanisms that balance immune cell responses in the intestine and to employ this knowledge in the design of successful new mucosal vaccines and immune-therapies.